Long-read sequencing reveals extensive gut phageome structural variations driven by genetic exchange with bacterial hosts

Genetic variations are instrumental for unraveling phage evolution and deciphering their functional implications. Here, we explore the underlying fine-scale genetic variations in the gut phageome, especially structural variations (SVs). By using virome-enriched long-read metagenomic sequencing across 91 individuals, we identified a total of 14,438 nonredundant phage SVs and revealed their prevalence within the human gut phageome. These SVs are mainly enriched in genes involved in recombination, DNA methylation, and antibiotic resistance. Notably, a substantial fraction of phage SV sequences share close homology with bacterial fragments, with most SVs enriched for horizontal gene transfer (HGT) mechanism. Further investigations showed that these SV sequences were genetic exchanged between specific phage-bacteria pairs, particularly between phages and their respective bacterial hosts. Temperate phages exhibit a higher frequency of genetic exchange with bacterial chromosomes and then virulent phages. Collectively, our findings provide insights into the genetic landscape of the human gut phageome.

. Level 1 and level 2 functional categories of annotated phage genes, along with their corresponding searching keywords.

Fig. S2 .
Fig. S2.Distribution and characteristics of structural variations (SVs) in human gut phageome.(A) Histogram depicting the distribution of the SV counts carried by individual phage species.The dashed line represents the average number of SVs carried.(B) Relationship between the read coverage of each phage and SV density, categorized by the phage lifestyle.(C) Histogram depicting the distributions of the number of supporting reads for detected phage SVs.(D) Comparison of GC content of genes residing within phage SVs and conserved regions.

Fig. S4 .
Fig. S4.Distribution of sequence identity of GE-like phage SV sequences detected in CHGV-HQ genomes to bacterial fragments, stratified by aligned bacterial datasets: CHGB and HumGut.The SV sequences exhibiting 100% nucleotide identity to bacterial fragments indicates latest horizontal gene transfers.

Fig. S5 .
Fig. S5.The enrichment of genes related to genetic exchange in regions with noGE-like phage SVs and GE-like phage SVs, stratified by SV types.
Fig. S6.Phylogenetic trees of CRISPR-associated cas1 protein along with bacterial and phage homologs.Maximum likelihood phylogenies were generated in IQ-Tree using the LG+F+R5.

Fig. S7 .
Fig. S7.Pivotal roles of phage-host interactions in phage SV information.(A) The number of SVs per 1 Mb among phages with different host range.(B) Positive correlations between the number of shared SV sequences within phage-bacteria pairs and the strength of these phage-bacteria correlations.The green line represents the regression line with shaded region showing 95% confidence interval.

Fig. S9 .
Fig. S9.Enriched functions in SVs linked to phages with different lifestyles.(A) The enriched functional categories of phage SVs for temperate and virulent phages, respectively (One-sided Fisher's exact test, false discovery rate, or FDR < 0.05).(B) The distribution of each functional category in the SVs associated with temperate and virulent phages.

Fig. S11 .
Fig. S11.Distribution of sequence identity of GE-like SV sequences detected in IMG/VR viral genomes to bacterial fragments, stratified by aligned bacterial datasets: CHGB and HumGut.The SV sequences exhibiting 100% nucleotide identity to bacterial fragments indicates latest horizontal gene transfers.

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Fig. S12 .
Fig. S12.Phage-bacteria interaction network constructed from the IMG/VR virus dataset with edges indicating that there are shared GE-like phage SVs between phages and bacteria.